Estrous (menstrual) cycle and estrogens influence pain sensitivity and analgesic effectiveness of -opioid re- ceptor (MOR)-selective opioids, e.g., our recent finding that the analgesia elicited in female rats by the in- trathecal (i.t.) application of the endogenous MOR ligand endomorphin 2 (EM2) is dampened during diestrus but robust and comparable to that of males during proestrus. Uncovering the molecular bases for clamped spi- nal EM2 analgesia will reveal novel molecular targets for drug interventions that disinhibit, and thus harness, endogenous EM2 analgesia, lessening the use of prescription opioids, and thereby their abuse. The organizing rubric generating Aims is that the phasic nature of spinal EM2 analgesia over the estrous cycle results from the plasticity of interactions among spinal MOR, ?-opioid receptor (KOR), aromatase (Aro), membrane estrogen receptor ? (mER?), mGluRs and their associated signaling partners. The first three aims focus on the molecu- lar components (and their organization) that regulate i.t. EM2 analgesia. The fourth aim investigates transla- tional relevance of findings. Aim 1 tests the hypothesis that during diestrus, spinal mER?-mGluR1 signaling, via phospholipase C and ??arrestin, dampens spinal EM2 analgesia. Aim 2 tests the hypothesis that during proestrus, mER? and ??arrestin are no longer relevant to spinal EM2 analgesia; mGluR1 now organizes with mGluR2/3 to signal via c-Src, facilitating spinal dynorphin release, which enables robust spinal EM2 analgesia to emerge. Aim 3 tests the hypothesis that spinal cord contains a novel modulatory oligomer comprised of MOR, KOR, Aro (thus locally synthesized estrogens), mER?, mGluR1 and mGluR2/3 that subserves the dynam- ic modulation of spinal EM2 analgesia over the estrous cycle. Aim 3 also tests the hypothesis that variable ac- tivation of mER? (resulting from fluctuating spinal Aro activity, and thus the synthesis of estrogens immediately proximal to mER?) drives reorganization of the predicted oligomer in diestrus vs. proestrus. Aim 4 tests the hypothesis that interventions shown in Aim 1 to restore spinal EM2 analgesia (e.g., blockade of spinal mER?, mGluR1, or phospholipase C) will be antinociceptive in diestrous rats undergoing chronic pain, which should augment the endogenous spinal EM2 system. Conversely, Aim 4 will also test the hypothesis that interventions shown in Aim 2 to eradicate spinal EM2 analgesia (e.g., blockade of spinal mGluR2/3, c-Src) will be pronocicep- tive in proestrous rats experiencing chronic pain. Collectively, proposed research will provide a new paradigm and suggest new molecular targets (e.g., the predicted oligomer, spinal ER?/Aro) for developing novel phar- macotherapies for pain relief that harnesses the powerful endogenous EM2/MOR analgesic system. Therapies that harness endogenous opioids would lessen the need for their exogenous counterparts, thereby circumvent- ing prescription opioid abuse, which has reached epidemic proportions. Additionally, findings will provide in- sight into etiology of chronic pain in women, since dysfunction of the physiological switch from EM2 anal- gesically non-responsive to responsive states is likely to facilitate developing and/or sustaining chronic pain.